FIG. 6.
Cross-linking of Rpa2 to nascent DNA labeled in the RNA primer moiety. Protein-DNA conjugates radiolabeled from [α-32P]UTP were prepared and detected by autoradiography and Western blotting, and the cross-linked DNAs were sized, as detailed in Materials and Methods. (A) Autoradiogram of a preclearance (Prcl) immunoprecipitate (lane 1), an Rpa2 immunoprecipitate of a control replication mixture containing dTTP instead of BrdUTP (lane 2), or the standard mixture (lane 3). (B) Immunoblot probed with the anti-Rpa2 MAb. Shown are proteins extracted from a nonlabeled control (Cntrl) nuclear monolayer (lane 1), antibody alone (lane 2), a preclearance immunoprecipitate (lane 3), and Rpa2 immunoprecipitates from reaction mixtures without (lane 4) or with (lane 5) BrdUTP. (C) Nascent RNA-DNA released by proteolysis from photolabeled Rpa2 species of bands h and l (from panel A, lane 3) was resolved by gel electrophoresis (lanes 2 [band h] and 4 [band l]) along with respective unproteolyzed controls (lanes 1 and 3). h and l designate slow- and fast-migrating photolabeled Rpa2 species, respectively. H and L indicate heavy and light Ig chains, respectively. Prot.K proteinase K; M, protein size markers (in thousands). The arrow indicates the ECL signal of Rpa2, and the arrowhead indicates at the photolabeled derivative.